There has been a growing interest in the manufacture and use of microfluidic systems for the acquisition of chemical and biochemical information. Techniques commonly associated with the semiconductor electronics industry such as photolithography, wet chemical etching, etc., are being used in the fabrication of these microfluidic systems. The term xe2x80x9cmicrofluidicxe2x80x9d, refers to a system or device or xe2x80x9cchipxe2x80x9d having channels and chambers which are generally fabricated at the micron or submicron scale, e.g., having at least once cross-sectional dimension in the range of from about 0.1 xcexcm to about 500 xcexcm. Early discussion of the use of planar chip technology for the fabrication of microfluidic systems is provided in Manz et al., Trends in Anal. Chem. (1990) 10(5):144-149 and Manz et al., Adv. in Chromatog. (1993) 33:1-66, which describe the fabrication of such fluidic devices, and particularly microcapillary devices, in silicon and glass substrates.
Applications of microfluidic systems are myriad. For example, International Patent Appln. WO 96/04547 describes the use of microfluidic systems for capillary electrophoresis, liquid chromatography, flow injection analysis, and chemical reaction and synthesis. U.S. Pat. No. 5,942,443 entitled xe2x80x9cHIGH THROUGHPUT SCREENING ASSAY SYSTEMS IN MICROFLUIDIC DEVICESxe2x80x9d, issued on Aug. 24, 1999 discloses wide ranging applications of microfluidic systems in rapidly assaying large numbers of compounds for their effects on chemical, and preferably biochemical systems. Biochemical systems include the full range of catabolic and anabolic reactions which occur in living systems including enzymatic, binding, signaling and other reactions. Biochemical systems of particular interest include receptor-ligand interactions, enzyme-substrate interactions, cellular signaling pathways, transport reactions involving model barrier systems (e.g., cells or membrane fractions) for bio-availability screening, and a variety of other general systems.
One of the major advances in recent times has been the adaptation of microfluidic devices to the performance of the polymerase chain reaction (PCR) and other cyclic polymerase-mediated reactions. However, a significant problem faced by experimenters has been the control of process parameters such as temperature, reagent concentration, buffers, salts, other materials, and the like. In particular, PCR should be carried out at precisely controlled temperatures. For example, PCR is typically based on three discrete, multiply repeated steps: denaturation of a DNA template, annealing of a primer to the denatured DNA template, and extension of the primer with a polymerase to create a nucleic acid complementary to the template. The conditions under which these steps are performed are well established in the art. Each step has distinct temperature and time requirements typically as follows:
See Innis et al., PCR Protocols: A Guide to Methods and Applications (Academic Press, Inc.; 1990). Generally, microfluidic systems are well suited to the performance of PCR because they allow rapid temperature changes, quickly providing the correct temperature at each step. Further, because the microfluidic elements are extremely small in comparison to the mass of the substrate in which they are fabricated, the heat can be highly localized, e.g., it dissipates into and from the substrate before it affects other fluidic elements within the device.
In addition to efficient temperature control, an experimenter attempting to run PCR must overcome a second problem. Often the efficiency of amplification reactions is compromised by primer self-annealing (xe2x80x9cprimer dimerxe2x80x9d) as well as larger non specific side-reaction products arising due to inefficient reaction conditions. Such nonspecific fragments adversely affect the yield of desired specific fragments through competition with the specific target in the reaction. Furthermore, the nonspecific fragments that are approximately the same size as the specific product can cause erroneous interpretation of results. Researchers have concluded that these nonspecific side reaction products originate from DNA polymerase catalyzed extension of partially annealed 3xe2x80x2 ends of primers to nonspecific sites in complex DNA under ambient temperature conditions. Therefore, it appears that efficiencies of thermostable DNA polymerases are greatly reduced at ambient temperature relative to their peak efficiencies at higher temperatures.
A xe2x80x9cHot Startxe2x80x9d PCR method was developed as a means of reducing the amplification of nonspecific products. See, e.g., D""Aquila et al., (1991) Nucleic Acids Res. 19:37-49. In the earlier methods, one of the reaction components was withheld from the reaction until the reaction mixture was heated to a temperature greater than the annealing temperature, followed by the addition of the missing component. This approach causes the partially annealed 3xe2x80x2 primer ends to melt away from nonspecific sites, before they can be extended. Therefore, Hot Start PCR improves product yield and specificity. More recent approaches to xe2x80x9cHot Startxe2x80x9d PCR include the use of a heat-labile wax or jelly barrier that melts and permits mixing of aqueous components at an elevated temperature. Chou et al., (1992) Nucleic Acids Res, 20:1717-1723. A third method utilizes a monoclonal antibody for deactivating Taq DNA Polymerase at ambient temperature. When the reaction mixture is heated to the denaturation temperature, the deactivation of the polymerase is reversed thereby facilitating amplification of specific targets. Kellogg et al., (1994) Biotechniques 16: 1134-1137. Although all of the above described methods are a significant improvement over simple PCR, a common problem associated with all these methods is that these methods are cumbersome to use and time consuming when working with multiple samples.
For the foregoing reasons, there is a need for efficient methods, devices and systems for performing temperature dependent reactions, such as hot start PCR, on multiple sample targets. The present invention satisfies this and other needs.
The present invention is directed to microfluidic systems including methods and devices for performing temperature mediated reactions in a precise and efficient manner.
Generally, the present invention is directed to microfluidic devices and methods of using the same, wherein the devices incorporate improved channel and reservoir configurations such that reaction components of a temperature mediated reaction are heated in a region of the device, while additional reaction components are added into the heated reaction mixture from a separate source, e.g., through a side channel.
In a first aspect, the present invention provides a microfluidic device that comprises a body structure having at least one microchannel with a heating region. A plurality of electrical access channels having first and second ends intersect the at least one microchannel at the first end. The electrical access channels are in fluid communication with partially filled reservoirs at the second end.
In a related aspect, the present invention provides a microfluidic device that comprises a body structure having a microchannel with a heating region, wherein the heating region has a first reaction component or components disposed in it but not a second reaction component. The heating region of the microchannel has a first electrical resistance. Two electrical access channels having a second electrical resistance, which is lower than the first electrical resistance, are in fluid communication with the microchannel. A source of a second reaction component is in fluid communication and intersects the heating region of the microchannel at a first intersection. The electrical access channels intersect the reaction channel on different sides of the first intersection. For example, in a device used for Hot Start PCR, the heating region of the device has primers but no polymerase disposed in it and the polymerase enzyme is introduced into the reaction mixture via a side channel which intersects the microchannel at a first intersection.
In another aspect, the present invention provides a microfluidic device described above, but comprising a source of reaction mixture slugs, in fluid communication with the reaction channel.
In a yet another aspect, the present invention employs a material transport system for delivering the polymerase enzyme and the reaction mixture into the reaction channel.
The present invention also provides methods for performing temperature mediated reactions using the devices described herein, which method comprises loading a first component of a temperature mediated reaction in a heating region of the reaction channel of the device, wherein the reaction channel is heated by applying an electrical current and subsequently delivering a second component of the temperature mediated reaction into the reaction channel.
In a related aspect, the present invention also provides methods of performing a temperature mediated reaction as described above, on a series of reaction mixture slugs where the reaction mixture comprises a first component of the temperature mediated reaction.